Pneumolysin, a thiol-activated cytolytic toxin produced by many pneumococcal disease isolates, may be involved in the pathogenicity of pneumococcal infection by helping evade the host defense mechanisms. We are studing the group 19 pneumolysins and cloning the ply genes to examine the relationship of pneumolysin to virulence. Isolation and molecular cloning of the ply gene from group 19 chromosomal DNA were performed, using cloned ply genes from two different pneumococcal strains as probes. Types 19F, 19A, 19B, and 19C chromosomal DNA exhibited two bands in the autoradiography after treatment of the DNA with various enzymes, includeing EcoRI and Hind III. In contrast, these DNA samples showed one band, when treated with ClaI, yielding a main fragment of approximately 5 kb. Genomic DNA, containing the ply gene from 19F, 19A, 19B, and 19C strains, was analyzed by the polymerase chain reaction (PCR) technique. Oligonucleotides were prepared to the known DNA sequence of type 2. These oligonucleotides found homologous sequences and generated a 1.5 kb PCR product in all four type 19 isolates. They were also found to be similar in their restriction endonuclease digestion patterns. Thus, these group 19 ply genes showed similar characteristics.